The NLRP3 inflammasome is an important intracellular component of the mammalian innate immune system. Significant research over the last decade has vastly improved the knowledge of how the inflammasome responds to a large and diverse group of bacterial, viral, fungal, and endogenous pathogens. A key step in inflammasome activation is complex formation and oligomerization of three proteins. However, the exact oligomerization state and the relationship of this oligomerization state to inflammasome function have not yet been determined. The proposed research study described in this application will produce the three inflammasome proteins in a mammalian cell system and probe these questions. By investigating the structural mechanisms driving NLRP3 inflammasome activation, we hope to further the overall understanding of this critical component of the innate immune system and identify key elements that might be exploited for therapeutic modulation. New information on these mechanisms will have implications for the treatment of neurodegeneration, heart disease, and numerous inflammatory diseases. The proposed research study described in this application will express three recombinant inflammasome proteins in a mammalian cell system and investigate the oligomerization state of each protein. Furthermore, interactions between each protein partner and the whole ternary complex will be defined and their organization determined. Finally, we will elucidate how the higher-order oligomerization state impacts inflammasome function. The proposed studies will be a significant step forward in knowledge, will guide future investigations of this important inflammatory regulator, and will provide tremendous educational involvement and outreach for many students interested in biochemical, biophysical, and biomedical studies.